Tumoral and normal brain tissue extraction protocol for wide-scope screening of organic pollutants

Little is known about the presence of organic pollutants in human brain (and even less in brain tumors). In this regard, it is necessary to develop new analytical protocols capable of identifying a wide range of exogenous chemicals in this type of samples (by combining target, suspect and non-target strategies). These methodologies should be robust and simple. This is particularly challenging for solid samples, as reliable extraction and clean-up techniques should be combined to obtain an optimal result. Hence, the present study focuses on the development of an analytical methodology that allows the screening of a wide range of organic chemicals in brain and brain tumor samples. This protocol was based on a solid-liquid extraction based on bead beating, solid-phase extraction clean-up with multi-layer mixed-mode cartridges, reconstitution and LC—HRMS analysis. To evaluate the performance of the extraction methodology, a set of 66 chemicals (e.g., pharmaceuticals, biocides, or plasticizers, among others) with a wide range of physicochemical properties was employed. Quality control parameters (i.e., linear range, sensitivity, matrix effect (ME%), and recoveries (R%)) were calculated and satisfactory results were obtained for them (e.g., R% within 60–120% for 32 chemicals, or ME% higher than 50% (signal suppression) for 79% of the chemicals).

a r t i c l e i n f o Method name: Tumoral and normal brain tissue extraction protocol for wide-scope screening of organic pollutants.

Keywords:
Human tissue Non-target screening Suspect screening Bead beating LC-HRMS Human biomonitoring a b s t r a c t Little is known about the presence of organic pollutants in human brain (and even less in brain tumors). In this regard, it is necessary to develop new analytical protocols capable of identifying a wide range of exogenous chemicals in this type of samples (by combining target, suspect and non-target strategies). These methodologies should be robust and simple. This is particularly challenging for solid samples, as reliable extraction and clean-up techniques should be combined to obtain an optimal result. Hence, the present study focuses on the development of an analytical methodology that allows the screening of a wide range of organic chemicals in brain and brain tumor samples. This protocol was based on a solid-liquid extraction based on bead beating, solidphase extraction clean-up with multi-layer mixed-mode cartridges, reconstitution and LC -HRMS analysis. To evaluate the performance of the extraction methodology, a set of 66 chemicals (e.g., pharmaceuticals, biocides, or plasticizers, among others) with a wide range of physicochemical properties was employed. Quality control parameters (i.e., linear range, sensitivity, matrix effect (ME%), and recoveries (R%)) were calculated and satisfactory results were obtained for them (e.g., R% within 60-120% for 32 chemicals, or ME% higher than 50% (signal suppression) for 79% of the chemicals). Value of the Protocol:

Specifications
• Small amount of tissue required (from 50 mg ww) • Account for a wide spectrum of chemicals with different physicochemical properties, essential for nontarget analysis.
• Simple protocol

Background
The evaluation of the chemical exposome, defined as the total environmental exposure since conception hereinafter [ 1 , 2 ], is essential for assessing the potential health risk facing humanity [3] . Human biomonitoring (HBM) studies, making use of the last advances in high-resolution mass spectrometry (HRMS), have been usually applied to human biofluids such as urine, to some extent easy to sample [4][5][6] , also requiring lower sample treatment. However, there is a current need for analytical methodologies that are capable to evaluate the chemical exposome in some other overlooked tissues, such as brain and tumor biopsies. The focused analysis of these matrices will improve our understanding on how organic pollutants enter and interact with brain tissues.

Objective
The goal of the present study was to validate an analytical methodology to perform wide-scope target, suspect or non-target screenings of organic chemicals in brain samples from autopsies and tumor biopsies by liquid chromatography coupled to highresolution mass spectrometry (LC -HRMS). A set of 66 chemicals were selected and used to evaluate the methodology. The selection was done based on diverse physicochemical properties (LogP between − 0.2 and 6.3), presence of diverse heteroatoms (including S, P, Cl, Br, F…) and comprising different chemical classes (e.g., pharmaceuticals, perfluoroalkyl and polyfluoroalkyl substances (PFAS), biocides, UV-Filters, plastic additives, personal care products (PCPs), food related chemicals, anticorrosion agents, and flame retardants).

Sampling campaign
Brain human tissue was obtained from HUB-IDIBELL Pathology Biobank and autopsies carried out at the Pere Virgili Health Research Institute (IISPV), Reus (Tarragona), during 2022. All samples were maintained at − 80 °C until sample treatment and a pool of them ( n = 5) was used for method validation.

Sample treatment
The sample treatment procedure consisted of three different steps, based on solid-liquid extraction (SLE), solid-phase extraction clean-up (SPE) and reconstitution for LC -HRMS analysis. It is based in a previous methodology for the detection of emerging contaminants in biota described elsewhere [7] .
I. Solid-liquid extraction (SLE) 1. Fill extraction tubes with 1 g of zirconium beads. 2. Add 100-150 mg wet weight (ww) (weight and note exact mass) of the sample into the extraction tube. Minimum recommended mass is 50 mg. 3. Add surrogate standards solution (clothianidin-d3) to achieve a 50 μg L − 1 in vial (in the final extract). The final volume extract (in μL) will be adjusted to the initial mass for each brain tissue (in mg ww). Allow evaporation of solvent (60 min, room temperature) so the standards will be better permeated into the matrix. 4. Add 1 mL of the extractant. (See Note #1). 5. SLE is now performed by bead beating, using FastPrep-24 5 G instrument (total time: 30 s, power: 5.5). 6. Centrifuge the samples (11.000 g, 10 min). 7. Transfer the supernatant into a labelled glass tube. These glass tubes require a volume capacity higher than 3 mL. 8. Repeat steps 4-7 twice. All supernatant extracts from the same sample must be collected in the same glass tube. 9. Eliminate the excess of organic solvent by evaporation using a N 2 evaporator until half of the volume (1.5 mL aprox.). Select a flow that slightly breaks the surface tension of the sample. 10. Transfer the extract to a glass bottle ( > 100 mL). To completely transfer sample extract, clean the glass tubes with 3 mL of water, vortex it and add it again to the glass bottle (three times). 11. Add water up to 100 mL and adjust pH to 6 To control the instrument performance, isotopically labelled internal standards (IS) may be added also the injection day. These IS should be different from those previously added as surrogate.
Mix 118 mL of S1 with 82 mL of S2 and mix carefully. Then, add 200 mL volume of S1:S2 (59:41) mixture to 200 mL acetonitrile. Store the excess of the solutions in the fridge.
Note #2: Homemade cartridges contained: 0.2 g of Sepra ZT, 0.1 g Sepra ZTL-WCX, 0.1 g Sepra ZTL-WAX and 0.15 g of Isolute ENV + from Biotage. The empty cartridge is filled with a frit, then 0.2 g of Sepra ZT, another frit, 0.35 g of a mix of the rest of the sorbents and the last frit. For more details see Gago-Ferrero et al. [8] Note #3: Mixture A: Methanol (47% of the total volume), ethyl acetate (47% of the total volume),) and 32% ammonia solution (6% of the total volume).

LC -HRMS analysis
Instrumental analysis was performed in an Acquity UHPLC system (Waters, Milford, USA) coupled to a Q-Exactive Orbitrap mass analyser (Thermo Fisher Scientific, Dreieich, Germany) by means of an electrospray ionization interface (ESI) in positive (ESI + ) and negative (ESI-) modes. The chromatography and mass spectrometry parameters are described as follows: The instrument worked on data independent acquisition (DIA) mode. It consisted of a full scan with low collision energy and a full scan with a high collision energy (25 eV), in a mass-to-charge ratio ( m/z ) range from 67 to 1000 and resolving power of 60,000.

Quality assurance and quality control
Quality assurance and quality control (QA/QC) measures were applied to prevent from any contamination during sample treatments or instrumental analysis. Thus, glass material was rinsed with distilled water and acetone and heated (450 °C) before use.

Table 1
Results of method validation including linear range, limit of quantification (LOQ), limit of detection (LOD), recovery (R%) and matrix effect (ME%), as well as ionization mode (IM).  Recoveries were determined as the average at 5 and 20 (μg L − 1 ). c NA: data non-available as these chemicals were not spiked in the calibration curve in solvent. d Ionization mode positive ( + ), negative (-) or both ( + / -). e TP corresponds to Transformation Product.
Standards and internal standards were stored in amber glass vials at − 20 °C in the dark to avoid degradation. Procedural blanks were done following the same steps of the protocol to account for any contamination. The 10 μg L − 1 spiked point of the calibration curve of the pooled brain was injected every 10 injections to probe the repeatability of the signal. Methanol was injected every 10 injections to control possible carry-over issues. Internal standards were used as surrogate to control the sample treatment and some of them were added before injection to control the instrument performance and correct possible matrix effects.

Method validation
A set of 66 chemicals were employed to evaluate the performance of the methodology, and the results are summarized in Table 1 . These chemicals included pharmaceuticals, PFAS, biocides, UV-Filters, plastic additives, food related chemicals, anticorrosion agents, and flame retardants with different physicochemical properties (LogP in the range: − 0.2 to 6.3). The proposed protocol has been validated in brain samples with a composite sample of brain by using the aforementioned 66 target chemicals . Due to the wide range of physicochemical properties of the selected compounds, this method is appropriate for non-target analysis.The following quality control parameters were assessed: • Linearity. The linearity and linear range were evaluated with a calibration curve spiked at 0.1, 0.5, 1, 5, 10, 50, 100 μg L − 1 in the pooled sample of brain (final extract). In addition, the same calibration curve (0.1, 0.5, 1, 10, 50, 100 μg L − 1 ) was prepared in solvent (water/methanol, 1:1, v/v ) to evaluate matrix effect. • Matrix effect (ME%). The effect of the matrix was evaluated with the Eq. (1) .
Values under/above 100% meant signal suppression/enhancement, respectively. • Recovery (R%). A pooled sample was spiked at two concentration levels and analysed using the experimental protocol above.
Fortification levels were 5 and 20 μg L − 1 (theoretical concentration in vial, C theoretical ). Additionally, 3 samples were processed without standard addition as matrix blanks. Then, recoveries were calculated with the Eq. (2) for each fortification level: where |Peak Area Spiked| referred to the average peak area of the spiked pooled sample at each fortification level, and |Blanks| referred to the average peak area of the matrix blanks. The slope was obtained from the calibration curve in matrix. • Sensitivity. Method limits of quantification (LOQ) were estimated as the lowest observable peak in the calibration curve in matrix.
And the limits of detection (LODs) were considered as 3/10 of the LOQs.

Method performance
The validation results are summarized in Table 1 . Briefly, recoveries were satisfactory (in the range 60 -120%) for 32 chemicals. Additional 13 chemicals showed recoveries between 120 and 150% and the rest provided low recovery values (17-60%). Malathion showed particularly low recovery (2%) and other 7 chemicals were not properly recovered as presented a RSD > 40%. Regarding matrix effect, a high suppression was generally observed for all the chemicals, as expected for this complex matrix. Ninety-two percent of the tested chemicals showed signal suppression (79% showed suppression higher than 50%). However, the instrument response was good enough to achieve acceptable LOQs, with 59% of them ≤ 1 μg L − 1 , and almost 82% ≤ 5 μg L − 1 . All the chemicals presented a coefficient of determination (R 2 ) higher than 0.90 in matrix, and 32 of them, higher than 0.99. Thus, the presented methodology provided satisfactory results for a wide number of chemicals with diverse physicochemical properties in a very complex human tissue (and small quantities of sample). This good method performance was remarkable considering the complexity of the evaluated matrix, and it was attributed to the sample pretreatment simplicity and comprehensiveness, which allowed to avoid compound losses. However, a reduced number of analytes were poorly recovered, showing high limits of quantification and a high matrix effect.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.